1. Field of the Invention
The present invention relates to a novel GABAA receptor ε subunit (GABRE) and an alternative transcript thereof (ET2). More specifically, isolated nucleic acid molecules are provided encoding human GABRE and ET2 receptor subunits. ET2 and GABRE polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of ET2 and GABRE activities. Also provided are diagnostic methods for detecting aberrant GABRE and ET2 expression, as well as therapeutic methods for treating disorders involving ET2 and GABRE.
2. Related Art
In mammalian brain, synaptic inhibition of neuronal activity is mediated mainly by the action of the neurotransmitter γ-aminobutyric acid (GABA) at GABA-gated chloride ion channels. GABA is an amino acid derivative which is produced in neurons by the decarboxylation of glutamate.
GABAA receptor complexes are multi-subunit members of a ligand-gated ion channel gene superfamily. The binding of GABA to these complexes results in the opening of channels across the neuronal cell membrane through which anions flow down their concentration gradients. Chloride ions are the main anions conducted through these channels, and channel opening requires the binding of at least two GABA molecules per GABAA receptor complex. Thus, the binding of GABA to GABAA receptor complexes induces an increase in chloride ion conductance across the postsynaptic membrane which alters the chloride ion gradient resulting in both hyperpolarization of this membrane and inhibition of neuronal firing. After interaction with the extracellular regions of the GABAA receptor complexes, GABA is actively “pumped” back into the prejunctional neurons.
Neurotransmitter responsive ligand-gated ion channels are known to be major targets for psychoactive drugs (Sargeant, P., Annu. Rev. Neurosci. 16:403–443 (1993)). The GABAA receptor complex, for example, is the primary site of action for many of the drugs used to treat anxiety and seizure disorders such as the benzodiazepines, barbiturates and tranquilizers (e.g., Valium and Librium). These receptors serve as molecular control elements through which the levels of anxiety, vigilance, muscle tension, and epileptic activity, as well as other conditions, can be regulated by drug-induced modulations.
Heterogeneity with respect to subunit types which make up transmitter-gated ion channels has been shown for several multi-subunit receptor complexes. The alternative forms of these subunits are generally either encoded by distinct genes or arise due to alternative mRNA splicing (see generally Alberts, B. et al., Molecular Biology of the Cell, 3rd edition, Garland Publishing, Inc. (1994)).
The GABAA receptor complex is believed to assemble into a pentameric structure and, to date, at least fourteen mammalian GABAA receptor subunits have been identified (α1–6, β1–3, γ1–3, δ, ε). These receptor subunits fall within five families on the basis of amino acid homology (Whiting, P. J., et al., Int. Rev. Neurobiol. 38:95–138 (1995); Davies, P. A., et al., Nature 385:820–823 (1997)). While the majority of functional receptors contain α/β/γ or α/β/δ subunit combinations, additional subunits combinations can form GABA-activated chloride channels.
Variations in subunit combinations can result in different pharmacological properties being conferred upon the receptor complex (Davies, P. A. et al., Nature 385:820–823 (1997); reviewed in Whiting, P. J. et al., Int. Rev. Neurobiol. 38:95–138 (1995)). Thus, subpopulations of GABAA receptor complexes show differing sensitivity to GABA, steroid modulators, physiological regulation, disease processes, and pharmacological manipulation by drugs (e.g., benzodiazepines). The distributions of mRNAs encoding different GABAA receptor subunit polypeptides and their subtypes localized in the brain show significant regional variation consistent with pharmacological and biochemical evidence for receptor heterogeneity. Further, alterations in brain specific expression of GABAA receptor complex subunit polypeptides have been identified in human brain tissues of individuals suffering from alcoholism (Lewohl, J. et al., Brain Res. 751:102–112 (1997)).
Thus, the properties of the different subpopulations of GABAA receptor complexes are determined, at least in part, by the subunits expressed in the particular cell. The GABAA receptor complex ε subunit, for example, confers several unique pharmacological and biophysical properties when assembled in a GABAA receptor complex containing α and β subunits (Davies, P. A. et al., Nature 385:820–823 (1997)). Most notably, this subunit inhibits the ability of anesthetic agents to potentiate GABA-gated chloride currents.
While much work has been done with GABAA receptor complexes localized in the brain, functional GABAA receptors have been identified in rat heart (Matsuyama, S. et al., Am. J. Physiol. 264:1057–1061 (1993); McLemore, G. L. et al., Pharmacology 49:342–350 (1994)). Further, GABAA receptor subunit mRNA has also been identified in human pancreatic tissue (Yang, W. et al., FEBS Lett. 346:257–262 (1994)) and, more recently, in tissues of the human reproductive system (Hedblom, E. and Kirkness, E., J. Biol. Chem. 272:15346–15350 (1997)). Currently, little is known about the subunit composition or pharmacological characteristics of these extra-central nervous system GABAA receptor complexes.